EP3720951A1 - Compositions and methods for improving persistence of cells for adoptive transfer - Google Patents

Compositions and methods for improving persistence of cells for adoptive transfer

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Publication number
EP3720951A1
EP3720951A1 EP18830140.2A EP18830140A EP3720951A1 EP 3720951 A1 EP3720951 A1 EP 3720951A1 EP 18830140 A EP18830140 A EP 18830140A EP 3720951 A1 EP3720951 A1 EP 3720951A1
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Prior art keywords
cells
nkg2d
cell
inhibition
ligands
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English (en)
French (fr)
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David GILHAM
Simon BORNSCHEIN
Susanna RAITANO
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Celyad Oncology SA
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Celyad SA
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    • C12N5/0634—Cells from the blood or the immune system
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells

Definitions

  • the present application relates to the field of immunotherapy, more particularly to the manufacture of cells for adoptive cell therapy.
  • compositions and methods for improving in vivo persistence of cells intended for adoptive transfer This is achieved by making the cells less vulnerable to clearance caused by NK cells of the subject receiving the adoptive cell therapy.
  • ACT adoptive cell transfer
  • ACT refers to the transfer of cells, most typically immune cells, into a patient. These cells may have originated from the patient (autologous therapy) or from another individual (allogeneic therapy). The goal of the therapy is to improve immune functionality and characteristics, and in cancer immunotherapy, to raise an immune response against the cancer.
  • T cells are most often used for ACT, it is also applied using other immune cell types such as NK cells, lymphocytes (e.g. tumor-infiltrating lymphocytes (TILs)), dendritic cells and myeloid cells.
  • TILs tumor-infiltrating lymphocytes
  • lymphodepletion is often used as neoadjuvant therapy, to ensure there are no competing immune cells to repopulate the immune cell space. This is especially important for allogeneic therapy: as is the case with transplants, an immune response may be raised against non-self infused cells, both by the adaptive and the innate immune system. Sometimes myeloablation, high-dose chemotherapy that kills cells in the bone marrow, is also used. However, lymphodepletion or myeloablation are quite drastic measures that often result in severe side effects because of their effect on the immune system.
  • This objective is achieved by reducing the innate immune response to these cells, particularly the innate immune response involved in recognizing induced-self antigens.
  • a particularly well-known receptor involved in recognition of induced-self antigens is NKG2D. It was found that inactivation of one or more of the NKG2D ligands in immune cells intended for ACT at least partially masks these cells for the NK-mediated immune response. This results in reduced killing of the ACT cells and prolonged persistence in vivo.
  • engineered immune cells that contain an exogenous nucleic acid molecule and at least one of:
  • One or more inhibitors directed against one or more NKG2D ligands One or more inhibitors directed against one or more NKG2D ligands.
  • the NKG2D ligands are selected from: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. According to further specific embodiments, the NKG2D ligands are one or both selected from MICA and MICB.
  • the one or more inhibitors are selected from RNA inhibitors, antibodies and peptide inhibitors.
  • the genes that have been engineered to be inactivated are inactivated using Crispr/CAS, TALEN, ZFN, Meganucleases or MegaTAL technology.
  • the exogenous nucleic acid molecule encodes a chimeric antigen receptor or a TCR.
  • the chimeric antigen receptor is a NKG2D CAR.
  • methods are provided of rendering an immune cell less sensitive to clearance by NK cells comprising the inhibition of one or more NKG2D ligands in the immune cell.
  • the immune cell further comprises an exogenous nucleic acid molecule.
  • the exogenous nucleic acid molecule encodes a chimeric antigen receptor or a TCR.
  • the inhibition of one or more NKG2D ligands is through genetic inactivation of one or more NKG2D ligands (e.g. using Crispr/CAS, TALEN, ZFN, Meganucleases or MegaTAL technology) or by administering one or more NKG2D ligand inhibitors (e.g. a RNA inhibitor, an antibody or a peptide inhibitor).
  • the NKG2D ligand inhibitor is shRNA against one or more NKG2D ligands.
  • the NKG2D ligand inhibitor is shRNA against MICA and/or MICB.
  • the shRNA can be administered as such, or can be part of a viral vector (e.g. a retro- or lentiviral vector).
  • the one or more inhibitors directed against one or more NKG2D ligands are typically provided as an exogenous nucleic acid to be introduced in the engineered immune cell.
  • the inhibition of NKG2D ligands i.e. the engineering to inactivate the genes, or the addition of one or more inhibitors
  • the manufacturing of these cells does not involve the administration step.
  • the engineered immune cells or the compositions described herein are provided for use as a medicament. They are particularly suited for use in the treatment of cancer.
  • One or more inhibitors directed against one or more NKG2D ligands to a subject in need thereof.
  • the methods for treatment can be autologous methods (the subject receives cells that originated from his or her body) or can be allogeneic methods (the immune cells are derived from a donor that is not the subject).
  • the inhibition of NKG2D ligands i.e. the engineering to inactivate the genes, or the addition of one or more inhibitors
  • continues while the drug is administered to the patient e.g. because the ligand is knocked out, or e.g. because shRNA is introduced that is constitutively expressed).
  • Figure 8 Survival curve of a mouse AML model treated with mock cells or NKR2 with and without shRNA against MICA/B.
  • Figure 9 Inhibition of MICA protein by Crispr/Cas compared to control or shRNA in CD4 and CD8 T cells.
  • immune cells refers to cells that are part of the immune system (which can be either the adaptive or the innate immune system).
  • Particularly envisaged immune cells include white blood cells (leukocytes), including lymphocytes, monocytes, macrophages and dendritic cells.
  • Particularly envisaged lymphocytes include T cells, NK cells and B cells, most particularly envisaged are T cells.
  • Immune cells as used herein are typically immune cells that are manufactured for adoptive cell transfer (either autologous transfer or allogeneic transfer). In the context of adoptive transfer, note that immune cells will typically be primary cells (i.e. cells isolated directly from human or animal tissue, and not or only briefly cultured), and not cell lines (i.e.
  • the immune cell is a primary cell. According to alternative specific embodiments, the immune cell is not a cell from a cell line.
  • exogenous nucleic acid molecule refers to a nucleic acid molecule that has been introduced in the immune cell, typically through transduction or transfection.
  • endogenous refers to any factor or material that is present and active in an individual living cell and that originated from inside that cell (and that are thus typically also manufactured in a non-transduced or non-transfected cell).
  • NKG2D ligand or the plural “NKG2D ligands” as used in the application refers to the human genes MICA (Gene ID: 100507436), MICB (Gene ID: 4277), ULBP1 (Gene ID: 80329), ULBP2 (Gene ID: 80328), ULBP3 (Gene ID: 79465), ULBP4 or RAET1E (Gene ID: 135250), ULBP5 or RAET1G (Gene ID: 353091), ULBP6 or RAET1L (Gene ID: 154064) and their gene products (or the relevant homolog when cells of other species are used).
  • gene products is at least RNA (transcribed from the gene) and protein (encoded by the NKG2D ligand gene, and translated from the transcribed RNA).
  • an "inhibitor directed against a NKG2D ligand” or "a NKG2D ligand inhibitor” as used herein refers to a molecule that prevents, inhibits or reduces signaling through the NKG2D ligand. Inhibition can occur at the DNA, RNA or protein level, e.g. through prevention of transcription or translation, through contact inhibition, competitive inhibition or other means.
  • the term "inhibition of one or more NKG2D ligands" as used in the application refers to interference with the function of the gene product of one or more of the NKG2D ligands, either at the DNA level (by inhibiting the formation of NKG2D ligand gene product, i.e. by preventing or interfering with transcription), at the RNA level (by neutralizing or destabilizing mRNA to prevent or interfere with translation) or at the protein level (by neutralizing or inhibiting the one or more NKG2D ligands, or by targeting nascent protein during the translation process).
  • Neutralizing at the protein level can be achieved at the cellular surface (e.g. by inhibiting receptor-ligand interaction) or before the protein is expressed at the surface (e.g. by retaining the protein in an intracellular organelle).
  • the ultimate functional effect of inhibition of one or more NKG2D ligands will be inhibition of NK cell cell activation through NKG2D-mediated signals.
  • Inhibition of one or more NKG2D ligands does not necessarily mean complete ablation of the NKG2D- induced signal, although this is envisaged as well. Particularly with antisense RNA and siRNA, but with antibodies as well, it is known that inhibition is often partial inhibition rather than complete inhibition. However, lowering functional NKG2D ligand levels may have a beneficial effect even when complete inhibition is not achieved as it lowers the chance of the immune cells being cleared.
  • the inhibition will result in a decrease of 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or up to 100% of the gene product of one or more of NKG2D ligands.
  • Methods of measuring the levels of NKG2D ligand gene product are known to the skilled person, and these can be measured before and after the addition of the inhibitor to assess the decrease in levels of functional gene product, or can be compared to suitable control cells where the ligands are not inhibited.
  • the inhibition may result in a decrease of 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or up to 100% of NK cell-mediated lysis as compared to cells in which no NKG2D ligand is inhibited.
  • a “chimeric antigen receptor” or “CAR” as used herein refers to a chimeric receptor (i.e. composed of parts from different sources) that has at least a binding moiety with a specificity for an antigen (which can e.g. be derived from an antibody or a receptor) and a signaling moiety that can transmit a signal in an immune cell (e.g. a CD3 zeta chain).
  • a “TCR” as used herein refers to a T cell receptor. In the context of adoptive cell transfer, this typically refers to an engineered TCR, i.e. a TCR that has been engineered to recognize a specific antigen, most typically a tumor antigen.
  • NK cells refers to the removal of cells from the circulation, tissue or body - most typically, it refers to the removal of the introduced immune cells, e.g. from the circulation.
  • “Clearance by NK cells” in this context means that the clearance is mediated by natural killer cells, NK cells typically clear or kill other cells by inducing lysis or apoptosis of these cells.
  • the present application is the first to show that immune cells in which NKG2D ligands have been inactivated or inhibited are less sensitive to clearance by NK cells and thus are more effective as a therapeutic as they remain active for longer. This is based on the finding that it appears the transduction and culture of immune cells (patient cells or donor cells) to make them suitable for adoptive cell transfer (i.e., the manufacturing process of ACT cells) also increases the presence of induced self antigens, including NKG2D ligands, and that this is effectively a cause of clearance from circulation by NK cells.
  • NKG2D is the best studied receptor involved in recognition of induced-self antigens, it is not the only NK receptor in this family that recognizes stress-induced ligands (or induced self antigens, or markers of the abnormal self, all used as equivalents herein).
  • Other natural killer cell receptors that are able to bind induced-self antigens are NKG2C, NKG2E, NKG2F, NKG2H (like NKG2D, all CD94 molecules) or Natural Cytotoxicity Receptors (NCR) such as NKp 46, NKp30 and NKp44, and it is envisaged that the methods and compositions can be used for such chimeric receptors as well, mutatis mutandis.
  • NKG2D is used in the application, this also applies to NKG2C, NKG2E, NKG2F, NKG2H, NKp46, NKp30 and NKp44.
  • the ligands for NKG2C, E, F and H are nonclassical MHC glycoproteins class I (HLA-E in human).
  • NK cells As these engineered cells are less sensitive to clearance, it is also an object of the invention to provide methods of rendering an immune cell less sensitive to clearance by NK cells, comprising the step of inhibition of one or more NKG2D ligands in the immune cell. This inhibition can be achieved through genetic inactivation, or by the presence of a NKG2D ligand inhibitor in the cell.
  • the clearance of NK cells is a process that happens in the body
  • the methods provided herein are in vitro or ex vivo methods. Indeed, the inhibition step (either through genetic engineering/inactivation or by the introduction of an inhibitory molecule) happens during the manufacturing process of said cells to make them suitable for ACT, and will be done outside a human body, i.e.
  • the methods described herein are applicable during manufacturing of immune cells.
  • manufacturing of immune cells occurs when cells are being prepared or cultured for adoptive transfer. This can be autologous adoptive transfer (a subject receives his own cells that have been modified and/or expanded), or allogeneic adoptive transfer (a subject receives cells from a different individual).
  • in vitro methods are provided for rendering an immune cell less sensitive to clearance by NK cells, and these methods do not encompass the administration to the patient.
  • the cells typically will further comprise an exogenous nucleic acid molecule.
  • the nucleic acid molecule encodes a chimeric antigen receptor (CAR) or a TCR.
  • This CAR or TCR can be directed against a suitable target. Most typically, the CAR or TCR will be directed against a tumor target.
  • the CAR or TCR can be directed e.g.
  • CD123 (IL3R alpha), CD133, CEA, CLD18 (claudin 18, splice variant 2), CLL1, cMET, CS1, EGFR, EGFRvlll, EpCAM, ErbB123, FAP (fibroblast activation protein), folate receptor alpha, GD2, GPC3, FIERI, HER2 (also Neu, ErbB2 or CD340), IL-1A, IL13R alpha 2 (CD213A2), kappa light chain, Ll-CAM, LeY, mesothelin, MUC-1, MUC16, NKG2D, NKp30, NKp44, NKp46, NY-ESOl, PD- 1, PDL-1, PIGF, PSCA, PSMA,
  • Inhibition can occur in multiple ways: contact inhibition (competitive inhibition or non-competitive inhibition), inhibition by interfering with ligand or receptor expression, interfering with ligand or receptor localization (e.g. preventing migration to cell surface), inhibition by binding to ligand or receptor or preventing interaction of both, and inhibition of downstream signaling, to name a few.
  • Permanent inhibition of the one or more of the NKG2D ligands is typically achieved by genetic knockdown. It has indeed been shown that gene editing is one potential method to specifically eliminate target antigen expression in the engineered T cell (Gomes-Silva D et al.. Blood, 2017).
  • functional inhibition can be achieved at three levels.
  • a "knock-out" can be a gene knockdown or the gene can be knocked out by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques known in the art, including, but not limited to, retroviral gene transfer.
  • engineered nucleases include, but are not limited to, meganucleases, zinc finger nucleases, TALENs, megaTALs and CRISPR nucleases.
  • Meganucleases found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) for making site-specific double strand breaks in nucleic acids. This makes them naturally very specific for a target sequence, and through mutagenesis and high throughput screening, hybrid meganuclease variants can be made that recognize unique sequences.
  • ZFNs and TALEN technology is based on a non specific DNA cutting enzyme, which can then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs).
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target desired DNA sequences, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
  • TALENs work similar to zinc fingers, but rely on transcription activator like effectors (TALEs) for DNA recognition. TALEs are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities.
  • MegaTALs are derived from the combination of two distinct classes of DNA targeting enzymes. Meganucleases (also referred to as homing endonucleases) are single peptide chains that have the advantage of both DNA recognition and nuclease functions in the same domain. However, meganuclease target recognition is difficult to modify, and they often have reduced specificity and lower on-target cleavage efficiency than other genome targeting endonucleases. Transcription activator-like (TAL) effectors are DNA recognizing proteins that have been linked to separate DNA endonuclease domains in order to achieve a targeted DNA double strand break. In contrast to meganucleases, TALs are easily engineered to target specific DNA sequences.
  • TAL Transcription activator-like effectors are DNA recognizing proteins that have been linked to separate DNA endonuclease domains in order to achieve a targeted DNA double strand break. In contrast to meganucleases, TALs are easily engineered to target specific DNA sequences.
  • TAL effectors each coupled to a non-specific DNA cleavage domain, in which DNA cleavage only occurs when both TAL effectors bind their respective sequences and the two endonuclease domains dimerize in order to cleave the DNA.
  • TAL effector nucleases can cause off -target activity, are much larger than meganucleases, and require the delivery of two separate proteins.
  • a megaTAL is the unification of a TAL effector with a meganuclease.
  • CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats / Crispr associated protein
  • Cas typically Cas9 nuclease complexed with a synthetic guide RNA (gRNA)
  • gRNA synthetic guide RNA
  • Genetic knockdown of one or more NKG2D ligand genes in the immune cells may mean inhibition of any combination of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6; and thus may mean knockout of one, two, three, four, five, six, seven or eight genes.
  • inhibition of one or more NKG2D ligands can also be achieved by (typically transient) inhibition using an inhibitor.
  • Inhibitors may act by inhibition of one or more NKG2D ligands on the immune cells, but also by inhibition of proper localization of the NKG2D ligands.
  • the timeframe of inhibition/introducing the inhibitor will typically coincide with the timeframe of manufacturing of the immune cells for ACT.
  • These manufacturing protocols may vary in number and order of steps, but they typically contain a transduction step (in which e.g.
  • a CAR is introduced in the isolated immune cells), an expansion step (in which the cells are cultured and increase in number) and a harvesting step (in which the cells are isolated and reformulated or concentrated, prior to administration to a patient or for (cryo)preservation).
  • the step of introducing the inhibitor (or doing the engineering for gene inactivation) will particularly coincide with the transduction step.
  • Transient gene inactivation may for instance be achieved through expression of antisense RNA in the immune cells, or by administering antisense RNA to said cells.
  • An antisense construct can be delivered, for example, as an expression plasmid, which, when transcribed in the cell, produces RNA that is complementary to at least a unique portion of the target mRNA (here mRNA of a NKG2D ligand).
  • a more rapid method for the inhibition of gene expression is based on the use of shorter antisense oligomers consisting of DNA, or other synthetic structural types such as phosphorothiates, 2 0 alkylribonucleotide chimeras, locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholinos.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • morpholinos With the exception of RNA oligomers, PNAs and morpholinos, all other antisense oligomers act in eukaryotic cells through the mechanism of RNase H-mediated target cleavage.
  • an antisense oligomer refers to an antisense molecule or anti-gene agent that comprises an oligomer of at least about 10 nucleotides in length. In embodiments an antisense oligomer comprises at least 15, 18 20, 25, 30, 35, 40, or 50 nucleotides. Antisense approaches involve the design of oligonucleotides (either DNA or RNA, or derivatives thereof) that are complementary to an mRNA encoded by polynucleotide sequences of FMR1.
  • Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. This effect is therefore stoichiometric. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense polynucleotide sequences, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense polynucleotide sequence. Generally, the longer the hybridizing polynucleotide sequence, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Oligomers that are complementary to the 5' end of the message, e.g., the 5' untranslated region (UTR) up to and including the AUG translation initiation codon, should work most efficiently at inhibiting translation.
  • UTR 5' untranslated region
  • oligomers complementary to either the 5', 3' UTRs, or non coding regions of the target gene could be used in an antisense approach to inhibit translation of said endogenous mRNA encoded by the target gene.
  • Oligomers complementary to the 5' UTR of said mRNA should include the complement of the AUG start codon.
  • Antisense oligomers complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense oligomers should be at least 10 nucleotides in length, and are preferably oligomers ranging from 15 to about 50 nucleotides in length. In certain embodiments, the oligomer is at least 15 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.
  • a related method uses ribozymes instead of antisense RNA.
  • Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties that can be designed to target specific RNA sequences. Successful target gene inactivation, including temporally and tissue-specific gene inactivation, using ribozymes has been reported in mouse, zebrafish and fruit flies.
  • RNA interference is a form of post-transcriptional gene silencing. The phenomenon of RNA interference was first observed and described in Caenorhabditis elegans where exogenous double-stranded RNA (dsRNA) was shown to specifically and potently disrupt the activity of genes containing homologous sequences through a mechanism that induces rapid degradation of the target RNA.
  • siRNAs small interfering RNAs
  • the siRNA typically comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson Crick base pairing interactions (hereinafter "base paired").
  • the sense strand comprises a nucleic acid sequence that is identical to a target sequence contained within the target mRNA.
  • the sense and antisense strands of the present siRNA can comprise two complementary, single stranded RNA molecules or can comprise a single molecule in which two complementary portions are base paired and are covalently linked by a single stranded "hairpin” area (often referred to as shRNA). These artificial RNA molecules with a tight hairpin turn are particularly envisaged for gene silencing and are included in the term siRNA.
  • isolated means altered or removed from the natural state through human intervention.
  • an siRNA naturally present in a living animal is not “isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.
  • the siRNAs of the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
  • the siRNA of the invention can also comprise a 3' overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3' end of an RNA strand.
  • the siRNA of the invention comprises at least one 3' overhang of from one to about six nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from one to about five nucleotides in length, more preferably from one to about four nucleotides in length, and particularly preferably from about one to about four nucleotides in length.
  • the length of the overhangs can be the same or different for each strand.
  • the 3' overhang is present on both strands of the siRNA, and is two nucleotides in length.
  • the 3' overhangs can also be stabilized against degradation.
  • the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • siRNAs can be obtained using a number of techniques known to those of skill in the art. For example, the siRNAs can be chemically synthesized or recombinantly produced using methods known in the art.
  • the siRNA of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, III., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
  • siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing siRNA of the invention from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
  • the siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly, e.g. in breast tissue or in neurons.
  • the siRNAs of the invention can also be expressed intracellularly from recombinant viral vectors.
  • the recombinant viral vectors comprise sequences encoding the siRNAs of the invention and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in the tissue where the tumour is localized.
  • an "effective amount" of the siRNA is an amount sufficient to cause RNAi mediated degradation of the target mRNA, or an amount sufficient to reduce NKG2D ligand-induced signaling in NK cells.
  • RNAi mediated degradation of the target mRNA can be detected by measuring levels of the target mRNA or protein in the cells of a subject, using standard techniques for isolating and quantifying mRNA or protein as described above.
  • morpholino antisense oligonucleotides in zebrafish and frogs overcome the limitations of RNase H -competent antisense oligonucleotides, which include numerous non-specific effects due to the non target-specific cleavage of other mRNA molecules caused by the low stringency requirements of RNase H. Morpholino oligomers therefore represent an important new class of antisense molecule. Oligomers of the invention may be synthesized by standard methods known in the art. As examples, phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res.
  • methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 7448-7451). Morpholino oligomers may be synthesized by the method of Summerton and Weller U.S. Pat. Nos. 5,217,866 and 5,185,444.
  • Inhibition can also be achieved by inhibitors at the protein level.
  • a typical example thereof are antibodies against one or more of the NKG2D ligands.
  • the term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against the NKG2D ligand or any functional derivative thereof, with said antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab')2, F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof.
  • These antibodies of the invention including specific polyclonal antisera prepared against the target protein or any functional derivative thereof, have no cross-reactivity to other proteins.
  • the monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat immunized against the target protein or any functional derivative thereof, and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the target protein or any functional derivative thereof which have been initially used for the immunization of the animals.
  • the monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
  • the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies.
  • Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in U.S. Pat. No. 5,545,806.
  • PBL peripheral blood lymphocytes
  • SCID severe combined immune deficiency
  • fragments derived from these monoclonal antibodies such as Fab, F(ab)'2 and scFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention.
  • Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases.
  • monoclonal antibodies, or fragments thereof can be modified for various uses.
  • the antibodies involved in the invention can be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • said antibodies against a target protein or a functional fragment thereof are derived from camels.
  • Camel antibodies are fully described in W094/25591, WO94/04678 and in WO97/49805.
  • inhibitors of NKG2D ligands at the protein level include, but are not limited to, peptide inhibitors of NKG2D ligands, peptide-aptamer (Tomai et al., J Biol Chem. 2006) inhibitors of NKG2D ligands, and protein interferors or Pept-lnsTM as described in W02007/071789 or WO2012/123419, incorporated herein by reference.
  • Another way of inhibition at the protein level is by interfering with the secretory transport, so that the ligands are not transported to the cell membrane. Typically, this is a temporary form of inhibition and normal cellular location can be restored when the appropriate signal is given to the cells, however, if no such signal is given, the inhibition will be permanent.
  • An exemplary method according to this principle is the RUSH (retention using selective hooks) system (Boncompain et al., Nature Methods 2012 and WO2010142785).
  • Small molecule inhibitors e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries.
  • NKG2D ligand inhibitors are typically selected from siRNA, antibodies, peptide inhibitors; more particularly they are selected from siRNA or antibodies; most particularly they are siRNA (such as e.g. shRNA) molecules against one or more NKG2D ligands.
  • immune cells are used for adoptive therapy and thus are envisaged for use in the methods described herein.
  • immune cells include, but are not limited to, T cells, NK cells, NKT cells, lymphocytes, dendritic cells, myeloid cells, stem cells or iPSCs.
  • the latter two are not immune cells as such, but can be used in adoptive cell transfer for immunotherapy (see e.g. Jiang et al., Cell Mol Immunol 2014; Themeli et al., Cell Stem Cell 2015).
  • manufacturing will entail a step of differentiation to immune cells prior to administration.
  • stem cells and iPSCs used in manufacturing of immune cells for adoptive transfer are considered as immune cells herein.
  • the stem cells envisaged in the methods do not involve a step of destruction of a human embryo.
  • the engineered immune cells described herein are provided for use as a medicament.
  • the engineered immune cells described herein are provided for use in the treatment of diseases selected from inflammatory disease, cancer, or infection (e.g. viral, bacterial, fungal infection).
  • diseases selected from inflammatory disease, cancer, or infection (e.g. viral, bacterial, fungal infection).
  • infection e.g. viral, bacterial, fungal infection.
  • the cells and compositions described herein are provided for use in the treatment of cancer.
  • cancers can be treated, including, but not limited to, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer and thyroid cancer; most particularly envisaged cancers include leukemia (including AML), multiple myeloma, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, and pancreatic cancer.
  • leukemia including AML
  • multiple myeloma bladder cancer
  • breast cancer colorectal cancer
  • pancreatic cancer pancreatic cancer
  • the immune cells are provided for use in treatment is equivalent as saying that methods of treating disease are provided, comprising a step of administering these immune cells to a subject in need thereof.
  • methods of treating inflammatory disease comprising administering the cells to a subject in need thereof.
  • methods of treating cancer comprising administering the cells to a subject in need thereof.
  • methods of treating infection comprising administering the cells to a subject in need thereof.
  • compositions of treating cancer in a subject in need thereof comprising a step of administering an engineered immune cell to said subject, the immune cell containing an exogenous nucleic acid molecule and at least one of:
  • One or more inhibitors directed against one or more NKG2D ligands One or more inhibitors directed against one or more NKG2D ligands.
  • exogenous nucleic acid molecule encodes a chimeric antigen receptor or a TCR.
  • Typical targets for such CARs or TCRs are listed above.
  • the immune cells may be autologous to the subject to which the cells are to be administered, or may be allogeneic, i.e. originating from a different subject.
  • Example 1 T cell activation induces specific NKG2D ligand expression.
  • This example demonstrates the generation of an engineered immune cell, more particularly a T cell, comprising a chimeric NKG2D receptor (i.e. an exogenous nucleic acid molecule encoding a chimeric antigen receptor) and one or more shRNAs directed against one or more NKG2D ligands.
  • a chimeric NKG2D receptor i.e. an exogenous nucleic acid molecule encoding a chimeric antigen receptor
  • shRNAs directed against one or more NKG2D ligands.
  • NKG2D stress-induced ligands
  • MICA/B and MICB were upregulated on the cell surface of CD4 and CD8 T cells, with expression peaking at day 2-4 after activation. Subsequently, expression declined until day 10. ULBP1 and ULBP2 were expressed at low levels, whereas ULBP2 was restricted to CD4+ T cells ( Figure 1). There was little evidence of expression of the other ligands on T cells.
  • Example 2 Co-expression of MICA/MICB targeting shRNA reduces NK-receptor mediated killing of T cells and improves tumor cell killing in vitro
  • Example 3 Inhibition of IMKG2D ligands increases persistence of CAR T cells, decrease tumor burden and prolong survival in vivo
  • NKG2D antigen receptor also termed NKR2
  • NKR2 chimeric NKG2D antigen receptor
  • the global aim of this study was to assess the long-term persistence of Chimeric Antigen Receptor (CAR) T cells in NOD SCID gamma-c-/- (Non-Obese Diabetic Severe Combined ImmunoDeficiency Gamma, NSG) mice after a single intravenous (IV) injection.
  • CAR Chimeric Antigen Receptor
  • NOD SCID gamma-c-/- mice Non-Obese Diabetic Severe Combined ImmunoDeficiency Gamma, NSG mice after a single intravenous (IV) injection.
  • the persistence in blood of seven different types of T-cell treatments generated from a unique donor was assessed.
  • NSG mice were irradiated within 24 hours before the IV injection of CAR T cells.
  • These highly immunodeficient mice lack mature T-cells, B-cells, natural killer (NK) cells and are also deficient in multiple cytokine signaling pathways, therefore allowing human cell engraftment.
  • mice The persistence of the seven different T-cell treatments was performed in seven groups of four mice (except for the group treated with Mock T cells composed of three mice) treated with a single IV injection of the relevant T cells (10xl0 6 cells/mouse) within 8 weeks post-injection.
  • One group of 3 mice was receive an injection of vehicle and was used as control, as well as mice injected with Mock T cells.
  • mice On Day -1, all the mice were irradiated. The mice were placed in the irradiator X-RAD320. They were irradiated, not anesthetized in their cage, by X-ray at 1.44Gy. The irradiation was performed at 70cm of the X-ray tube with 0.5Gy/min dose rate and a standard protocol of mice irradiation using the following parameters:
  • the X-vivo 15 medium (X-VIVO 15 without Gentamycin and Phenol Red) were preheated at +37°C in a water bath,
  • the X-vivo 15 medium was supplemented with 1% of Gentamycin concentrated at 50mg/ml and with 5% of Human male AB Serum heat inactivated (HS) (i.e. 10ml of Gentamycin and 50 ml of HS were added in a 1000ml bottle of X-vivo 15 medium),
  • HS Human male AB Serum heat inactivated
  • the vials containing frozen cells were transferred in a water bath at +37°C until a small fragment of ice remains,
  • the vials were decontaminated and transferred under the laminar hood, lml of pure cold HS were added drop by drop in each vial of T cells, 8ml x the number of thawed vials of pre-warmed complete X-vivo 15 medium were deposited in a 50ml Falcon tube (16ml for 2 vials of T cells),
  • the cells were transferred in these 50ml Falcon tubes and the cryovials were rinsed with lml of cell suspension in order to transfer the totality of T cells, -
  • the 50ml Falcon tubes were centrifuged at 400g for 5 minutes and then, the supernatants were discarded carefully,
  • the remaining thawed cells were used to perform the flow cytometry panel validation.
  • the vehicle, the test and reference items were administered by IV injection in one tail vein with disposable plastic syringes of lml and 26G needles. One syringe per group was used.
  • Whole blood (WB) of each mouse of all groups (Groups 1 to 8) was collected on Days 1, 6, 13, 20, 27, 34, 41, 48 and 55.
  • WB was collected by retro-orbital sinus on anesthetized mice (lsofluranel-3%), through capillary tubes.
  • Animals were euthanized under anesthesia (mix of Isoflurane and oxygen as a carrier gas) by cervical dislocation.
  • Flow cytometry analyses were performed on 30 samples of WB on Days 1, 6, 13, 20, 27, 34, 41 and 48 and on 29 samples on Day 55.
  • Human T cells were detected, on WB, after staining, using a combination of mAbs, described in Table 2, containing anti-human CD45 (hCD45), anti-hCD3, anti-hCD314, anti-hCD19, anti-hMICA/B and anti mouse (mCD45) mAb, in order to exclude murine cells.
  • NKG2D CAR T cells showed a persistence of 24 days in NSG mice.
  • Coexpression of MICA/B targeting shRNA #2 improved engraftment until day 41.
  • co-expression of shRNA #4 increased persistency of CAR-T cells in the peripheral blood by at least a factor of 2 until the end of the experiments in all animals.
  • AML Acute Myeloid Leukemia
  • cytotoxic chemotherapy achieves high remission rates, about 75% of patients will either not respond to or will relapse after initial therapy, and most patients will die of their disease.
  • NKR-2 CAR T cells against AML Antitumor efficacy of NKR-2 CAR T cells against AML was assessed in immunodeficient NOD SCID Gamma-c-/- (Non-Obese Diabetic Severe Combined ImmunoDeficiency Gamma, NSG) mice using the THP-l-luc-GFP cell line (acute monocytic leukemia - AML subtype 5) expressing luciferase (luc) and Green Fluorescent Protein (GFP).
  • THP-l-luc-GFP cell line acute monocytic leukemia - AML subtype 5
  • luc luciferase
  • GFP Green Fluorescent Protein
  • NKR-2 human T cells genetically modified with a retroviral vector coding for a chimeric receptor based on the NKG2D NK receptor generated with a culture process using a blocking mAb
  • NKR-2 shRNA T cells human T cells genetically modified with a retroviral vector coding for a chimeric receptor based on the NKG2D NK receptor and one short hairpin RNA (shRNA) candidate targeting 2 NKG2D ligands, MICA/B (either shRNA #2 or #4) was assessed in this mouse model.
  • shRNA short hairpin RNA
  • test items, reference item and vehicle were IV administrated 7 days after the tumor cell injection.
  • the antitumor efficacy of the different CAR-T cells was evaluated by in vivo bioluminescence imaging.
  • a visualization and quantification of the THP-l-luc-GFP cell proliferation and dissemination in the whole animal was performed 8 times within 8 weeks after their IV injection (on Days 4, 8, 13, 22, 29, 36, 43 and 57).
  • mice were IV injected with THP-l-luc-GFP cells (5x10 s cells/mouse) at day 0, followed at day 7 with the injection of the relevant vehicle, mock or CAR T cells.
  • NKR2 cells with shRNA against MICA/B appear to slow tumor growth, as evidenced by a reduced tumor burden, particularly in longer timeframes.
  • all mice treated with mock cells succumbed to the tumor by day 19.
  • mice treated with a CAR survived till day 30.
  • shRNA against NKG2D ligands did not extend survival of all mice beyond day 30, mice start to die significantly later than those injected with a CAR alone, indicating an increased benefit on survival of the shRNA. In a less aggressive tumor model or with repeated injections, this benefit may be further improved.
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